Craniofacial and long bone defects often exhibit delayed or non-union healing. While autogenous bone grafts and bone morphogenetic proteins are widely used to treat such severe injuries, these therapies have limitations. Therefore, a clinical need persists for the development of new methods to enhance bone regeneration. The Notch signaling pathway regulates osteoprogentor proliferation and osteoblast differentiation. Additionally, Notch signaling is an essential component for wound healing in many tissues, and Notch upregulation following injury has been shown to enhance regeneration of those tissues. Therefore, the long-term objectives of this study are to show that Notch signaling also regulates regeneration of craniofacial and long bone tissue, and that upregulation of Notch signaling through a clinically-applicable tissue engineering strategy can enhance craniofacial bone regeneration. The following three specific aims complement the NIDCR's strategic plan for 2009-2013 (draft) objective 1-5, which is to 'Facilitate reconstruction and regeneration of diseased or damaged oral and craniofacial tissues and organs through biological, bioengineering and biomaterials research approaches.' For the first specific aim we will characterize Notch signaling during calvarial defect and tibial bone fracture healing. Notch reporter mice will undergo a 3 mm diameter calvarial defect or bilateral tibial fractures. Mice will be sacrificed pre-injury and at 1, 2, 5, 10, 15, 20 and 40 days post-injury for qPCR and immunohistochemistry analysis. In specific aim 2, we will determine the critical importance of Notch signaling for successful calvarial and tibial bone regeneration. Transgenic mice with Cre-regulated expression of the canonical notch inhibitor dnMAML will undergo a 3 mm calvarial defect or bilateral tibial fractures and adeno-Cre will be locally administered tp permanently inhibit Notch signaling (adeno-LacZ control). Mice will be sacrificed at 5, 10, 20 and 40 days post-injury for |JCT, histology, and biomechanical testing. For the final specific aim we will develop a Notch tissue engineering therapy to enhance calvarial bone regeneration. As a proof of concept, we will administer adeno-NICD (adeno-LacZ control) to 3 mm calvarial defects in wild type mice to transiently upregulate Notch. We will then adsorb Notch ligand, Jagged-1 onto an osteoconductive biomaterial and implant these tissue engineered constructs in 3 mm calvarial defects. The purpose of this study is to 1) investigate the role(s) of the Notch signaling pathway in craniofacial bone healing, and 2) show that Notch manipulation can clinically enhance craniofacial bone regeneration. Public Health Relevance: Craniofacial and long bone defects often exhibit delayed or non-union healing. While autogenous bone grafts and bone morphogenetic proteins are widely used to treat such severe injuries, these therapies have limitations. Therefore, a clinical need persists for the development of new methods to enhance bone regeneration. The purpose of this study is to 1) investigate the role(s) of the Notch signaling pathway in craniofacial bone healing, and 2) show that Notch manipulation can clinically enhance craniofacial bone regeneration.